Journal: Nucleic Acids Research

Article Title: Formation and persistence of polyglutamine aggregates in mistranslating cells

doi: 10.1093/nar/gkab898

Figure Lengend Snippet: Selected observed peptides showing Ser mis-incorporation at Phe codons in mCherry

Article Snippet: Briefly, mCherry protein and wild-type or mistranslating tRNA Ser were co-expressed in N2a cells and mCherry protein was purified using RFP-trap agarose bead immunoprecipitation (Chromotek, Munich, Germany).

Techniques: Sequencing

Journal: Nucleic Acids Research

Article Title: Formation and persistence of polyglutamine aggregates in mistranslating cells

doi: 10.1093/nar/gkab898

Figure Lengend Snippet: Observed spectral counts for Ser or Phe incorporation at Phe codons in mCherry

Article Snippet: Briefly, mCherry protein and wild-type or mistranslating tRNA Ser were co-expressed in N2a cells and mCherry protein was purified using RFP-trap agarose bead immunoprecipitation (Chromotek, Munich, Germany).

Techniques:

Journal: Nucleic Acids Research

Article Title: Formation and persistence of polyglutamine aggregates in mistranslating cells

doi: 10.1093/nar/gkab898

Figure Lengend Snippet: Fluorescence and expression of mCherry protein in mistranslating cells. N2a cells were transfected with a plasmid encoding human tRNA Ser AGA or G35A variant tRNA Ser AAA ; or tRNA Ser CGA or the C34G, A36G variant tRNA Ser UGG and fluorescently dead GFP (S65F or P) fused to an active mCherry transfection marker. Fluorescence of cells was measured by fluorescence microscopy (RFP, ex 531 nm, em 593 nm). Each box represents data from 135 cells from three biological and nine technical replicates ( A ). Midline represents the median, boxes represent quartiles, and whiskers represent 1.5× the interquartile range. Representative images were captured under 20× magnification with phase or RFP settings ( B ). Cell lysates were harvested and western blotted for fluorescent protein expression with anti-GFP and anti-GAPDH antibodies as a loading control ( C ). Anti-GFP blots were quantitated in three biological replicates by densitometry normalized to GAPDH. Stars indicate P -values from independent sample t-tests (n.s. = no significant difference, * P < 0.05, ** P < 0.01, *** P < 0.001) and letters indicate significantly different groups determined by Tukey's Honestly Significant Different (HSD) test, where groups sharing a letter are not significantly different and groups not sharing a letter are significantly different (α = 0.05).

Article Snippet: Briefly, mCherry protein and wild-type or mistranslating tRNA Ser were co-expressed in N2a cells and mCherry protein was purified using RFP-trap agarose bead immunoprecipitation (Chromotek, Munich, Germany).

Techniques: Fluorescence, Expressing, Transfection, Plasmid Preparation, Variant Assay, Marker, Microscopy, Western Blot, Control

Journal: Nucleic Acids Research

Article Title: Formation and persistence of polyglutamine aggregates in mistranslating cells

doi: 10.1093/nar/gkab898

Figure Lengend Snippet: Protein turnover and clearance of PolyQ aggregates in mistranslating cells. N2a cells were transfected with a plasmid encoding human tRNA Ser AGA or G35A variant tRNA Ser AAA and HTTexon1 containing 23Q or 74Q fused to EGFP for 48 h before cycloheximide chase assays (A−D). Cells were treated with cycloheximide and/or MG132 and fluorescence was captured by live cell fluorescence microscopy. ( A ) Representative images of 23Q-EGFP/tRNA Ser AGA and 23Q-EGFP/tRNA Ser AAA expressing cells at the indicated timepoints. Fluorescence was quantitated in ImageJ (see supplemental methods). Average rate of protein decay ( B ) is shown as a change in fluorescence normalized to initial cell area ( C , D ) (see supplemental information). ( E ) PC12 cells with genomically integrated HTT-exon1 74Q-EGFP under a doxycycline (Dox)-inducible promoter were transfected with a plasmid encoding human tRNA Ser AGA or G35A variant tRNA Ser AAA and mCherry transfection marker. HTT-exon1 expression was induced with doxycycline 48 h post-transfection and cells were imaged daily by fluorescence microscopy. After 96 h, Dox was removed and daily imaging was resumed for 72 h. Aggregate counting is described in methods (see ). Error bars represent the mean ±1 standard deviation of at least three biological replicates and nine technical replicates each. Stars indicate P -values from independent sample t-tests (n.s. = no significant difference, * P < 0.05, ** P < 0.01, *** P < 0.001) and letters indicate significantly different groups determined by Tukey's HSD test (α = 0.05).

Article Snippet: Briefly, mCherry protein and wild-type or mistranslating tRNA Ser were co-expressed in N2a cells and mCherry protein was purified using RFP-trap agarose bead immunoprecipitation (Chromotek, Munich, Germany).

Techniques: Transfection, Plasmid Preparation, Variant Assay, Fluorescence, Microscopy, Expressing, Marker, Imaging, Standard Deviation

Journal: bioRxiv

Article Title: Glycine acylation and trafficking of a new class of bacterial lipoprotein by a composite secretion system

doi: 10.1101/2020.09.23.310854

Figure Lengend Snippet: Unique Aat amino acid sequences were detected using PSI-BLAST. These sequences were using to identify the strains that encoded them in the NCBI identical protein groups. As the aat genes were present on contigs of whole genome sequencing projects it was not possible to assess if a strain encoded a gene on the chromosome or plasmid. Instead contigs were used to identify complete the complete Aat system. This analysis does not include strains that might encode the aat genes or aap/cexE on a separate genomic element. However, a total of 827 complete Aat systems were identified in the same nucleotide accession. The positions of these genes were used to assess the organisation of the aat operon. From this assessment five different classes of aat operon organisation were defined.

Article Snippet: The cexE -mCherry fusion and the aatD gene from ETEC H10407 were synthesised by GenScript.

Techniques: Sequencing, Plasmid Preparation

Journal: bioRxiv

Article Title: Glycine acylation and trafficking of a new class of bacterial lipoprotein by a composite secretion system

doi: 10.1101/2020.09.23.310854

Figure Lengend Snippet: (A) Culture supernatant of ETEC H10407 and aat mutants harbouring pCfaD grown in LB supplemented with L-arabinose. Cells were removed from the culture supernatant and remaining protein precipitated with trichloroacetic acid. Protein samples were separated by tris-tricine SDS-PAGE and stained with Coomassie or transferred to nitrocellulose for western blotting with polyclonal antibodies against CexE. (B) Whole cell lysates of the aat mutants and parental strain separated by tris-tricine SDS-PAGE. (C) Whole cell lysates of ETEC H10407 grown in LB with or without azide. (D) Whole cell lysates of EAEC 042, aap, aatD and aatD complemented strains grown in DMEM high glucose. The positions of uCexE, mCexE, uAap, mAap, and preCexE are indicated as appropriate. CexE and Aap were detected by western blotting (α-CexE and α-Aap, respectively) and RNA polymerase (α-RNAP) was included as a loading control where appropriate.

Article Snippet: The cexE -mCherry fusion and the aatD gene from ETEC H10407 were synthesised by GenScript.

Techniques: SDS Page, Staining, Western Blot, Control

Journal: bioRxiv

Article Title: Glycine acylation and trafficking of a new class of bacterial lipoprotein by a composite secretion system

doi: 10.1101/2020.09.23.310854

Figure Lengend Snippet: His tagged CexE was isolated from cexE or cexE aatD double mutants harbouring pACYC- cexE -6His grown in the presence or absence of 17-ODYA and separated by SDS-PAGE. An azide linked Alexa Fluor 488 was conjugated to the alkyne moiety present in 17-ODYA by CuAAC. The incorporation of 17-ODYA into the target protein was detected by fluorescence (green bands) and the image was overlaid on the image of the SDS-PAGE gel. A known lipoprotein YraP was used as a positive control.

Article Snippet: The cexE -mCherry fusion and the aatD gene from ETEC H10407 were synthesised by GenScript.

Techniques: Isolation, SDS Page, Fluorescence, Positive Control

Journal: bioRxiv

Article Title: Glycine acylation and trafficking of a new class of bacterial lipoprotein by a composite secretion system

doi: 10.1101/2020.09.23.310854

Figure Lengend Snippet: The membrane fraction of ETEC H10407 and aat mutant harbouring pCfaD were separated by SDS-PAGE. Transferred to nitrocellulose and probed for the cytoplasmic protein RNAP, lipoprotein NlpE and CexE using protein specific antibodies.

Article Snippet: The cexE -mCherry fusion and the aatD gene from ETEC H10407 were synthesised by GenScript.

Techniques: Membrane, Mutagenesis, SDS Page

Journal: bioRxiv

Article Title: Glycine acylation and trafficking of a new class of bacterial lipoprotein by a composite secretion system

doi: 10.1101/2020.09.23.310854

Figure Lengend Snippet: E. coli BL21(DE3) was transformed with pET26b, or pET26b- cexE and pACYCDuet-1 or pACYC- aatD . Cultures were grown in LB and protein production was stimulated with IPTG. Whole cell lysate samples were taken and separated by tris-tricine SDS-PAGE. CexE was detected using a polyclonal antibody. RNAP was used as a loading control. The position of mCexE and uCexE are indicated.

Article Snippet: The cexE -mCherry fusion and the aatD gene from ETEC H10407 were synthesised by GenScript.

Techniques: Transformation Assay, SDS Page, Control

Journal: bioRxiv

Article Title: Glycine acylation and trafficking of a new class of bacterial lipoprotein by a composite secretion system

doi: 10.1101/2020.09.23.310854

Figure Lengend Snippet: (A) AatD from EAEC 042 and ETEC H10407 were aligned with Lnt from MG1655. AatD sequences identified by PSI-BLAST were aligned and used to create a Weblogo. The catalytic residues of the C-N hydrolase family are highlighted. (B) The structure of Lnt compared to the predicted structure of ETEC H10407 AatD. The catalytic residues of Lnt and AatD are highlighted in orange. (C) Magnified view of the catalytic site of Lnt with the predicted structure of AatD superimposed. (D) Residues are numbered as they appear in Lnt. E. coli BL21(DE3) was producing CexE in the presence of each of the four single site substitution mutants of AatD. CexE was detected using a polyclonal antibody.

Article Snippet: The cexE -mCherry fusion and the aatD gene from ETEC H10407 were synthesised by GenScript.

Techniques:

Journal: bioRxiv

Article Title: Glycine acylation and trafficking of a new class of bacterial lipoprotein by a composite secretion system

doi: 10.1101/2020.09.23.310854

Figure Lengend Snippet: (A) SignalP result of CexE sequence from ETEC H10407 (E3PPH2_ECOH1) (B) Weblogo of the 5 N-terminal residues of Aap and CexE sequences post Sec signal sequence cleavage. (C) ETEC H10407 cexE mutants transformed with pACYC184 (Δ cexE ) or pACYC- cexE -6His with either the wild-type sequence (WT cexE ) or one of the first five amino acids mutated to glutamic acid. CexE was detected by polyclonal antibodies and RNAP was used as a loading control. The percentage of mCexE was determined from 3 biological replicates.

Article Snippet: The cexE -mCherry fusion and the aatD gene from ETEC H10407 were synthesised by GenScript.

Techniques: Sequencing, Transformation Assay, Control

Journal: bioRxiv

Article Title: Glycine acylation and trafficking of a new class of bacterial lipoprotein by a composite secretion system

doi: 10.1101/2020.09.23.310854

Figure Lengend Snippet: Aap and CexE sequences were aligned using ClustalO. The alignment was then placed into WebLogo.

Article Snippet: The cexE -mCherry fusion and the aatD gene from ETEC H10407 were synthesised by GenScript.

Techniques:

Journal: bioRxiv

Article Title: Glycine acylation and trafficking of a new class of bacterial lipoprotein by a composite secretion system

doi: 10.1101/2020.09.23.310854

Figure Lengend Snippet: (A) CexE-6His was isolated from a cexE mutant and a cexE aatD double mutant and subjected to LC-MS/MS. CexE and pro-CexE were trypsinated and separated by HPLC. (B) The indicated peak was subjected to MS/MS to identify the amino acid sequence.

Article Snippet: The cexE -mCherry fusion and the aatD gene from ETEC H10407 were synthesised by GenScript.

Techniques: Isolation, Mutagenesis, Liquid Chromatography with Mass Spectroscopy, Tandem Mass Spectroscopy, Sequencing

Journal: bioRxiv

Article Title: Glycine acylation and trafficking of a new class of bacterial lipoprotein by a composite secretion system

doi: 10.1101/2020.09.23.310854

Figure Lengend Snippet: Heterologous acylation by AatD plasmid encoding mCherry with the CexE signal sequence followed by none, one (G), two (GG) or three (GGG) glycine residues post signal sequence was produced in an ETEC H10407 cexE mutant grown in the presence of 17-ODYA. The mCherry proteins were isolated using a C-terminal 8 His tag and azide linked Cy5 was incorporated into mCherry proteins using CuAAC. The acylation of mCherry was detected using fluorescence.

Article Snippet: The cexE -mCherry fusion and the aatD gene from ETEC H10407 were synthesised by GenScript.

Techniques: Plasmid Preparation, Sequencing, Produced, Mutagenesis, Isolation, Fluorescence

Journal: PLOS Biology

Article Title: Phosphoinositide species and filamentous actin formation mediate engulfment by senescent tumor cells

doi: 10.1371/journal.pbio.3001858

Figure Lengend Snippet: MCF-7 cell lines expressing biosensors that detect indicated PI species were treated with 250 nM doxorubicin for 24 hours, washed, and plated on untreated mCherry-MCF-7 “prey” cells. DOXO-NT cultures were imaged over days 3–8. Time course live-cell imaging of senescent MCF-7 cells that express ( A ) PLCD1-GFP marking PI(4,5)P2; ( B ) P4M-SidMx2-GFP marking PI(4)P; ( C ) 2xFYVE-GFP marking PI(3)P; ( D ) TAPP1-GFP marking PI(3,4)P2; ( E ) BTK-GFP marking PI(3,4,5)P3; ( F ) PLCD1(R40L)-GFP mutant that does not bind PI species (Negative Ctrl), throughout the entire process of engulfing mCherry-MCF-7 cells. Scale bar = 100 μm.

Article Snippet: 2xFYVE-domain-mCherry fusion protein is lentiviral plasmid from VectorBuilder.

Techniques: Expressing, Live Cell Imaging, Mutagenesis

Journal: PLOS Biology

Article Title: Phosphoinositide species and filamentous actin formation mediate engulfment by senescent tumor cells

doi: 10.1371/journal.pbio.3001858

Figure Lengend Snippet: (A ) Axial slices along the Z-axis and volume view reconstructions of a senescent MCF-7 cell expressing 2xFYVE-GFP and an mCherry expressing MCF-7 prey cell in a DOXO-NT culture. Top panel scale bar = 10 μm; bottom panel scale bar = 1 μm. ( B , C ) Representative image of a 2xFYVE-GFP cell engulfing a NIR-MCF-7 cell. Pixel intensity was determined across the distance of the yellow line and plotted in ( C ) for 5 individual cells. Scale bar = 60 μm. Underlying data can be found at . ( D ) Five 2xFYVE-GFP-MCF-7 predator cells were followed during engulfment of pHrodo red stained prey cells, and 2xFYVE-GFP intensity was measured at cytoplasm, membrane, and ring (as depicted in ) and pHrodo intensity was measured (right). The right graph shows the ratio of 2xFYVE-GFP intensity of the ring:cytoplasm (light green) over multiple hours surrounding pHrodo intensity increase for 5 cells. Error bars represent SEM. Underlying data can be found at . ( D ) Representative images of one 2xFYVE-GFP-MCF-7 predator cell engulfing pHrodo-labeled MCF-7 prey used for Fig 4D (right). ROIs used for quantification are shown in top panel. Scale bar = 100 μm. ( E ) Time course of a 2xFYVE-GFP-MCF-7 cell engulfing an MCF-7-mCherry prey cell. Dashed arrows indicate predator/prey contact; closed arrows indicate the overtopped prey cell with 2xFYVE concentration; open arrows indicate the prey cell no longer overtopped mCherry prey cell. Scale bar = 50 μm. ( F ) Axial slices along the Z-axis and volume view reconstructions of senescent 2xFYVE-GFP-MCF-7 predator cells engulfing mCherry-MCF-7 prey cells. Leftmost image shows the bottom axial slice. Closed arrows indicate 2xFYVE-GFP concentration at the prey cell. Scale bar = 10 μm.

Article Snippet: 2xFYVE-domain-mCherry fusion protein is lentiviral plasmid from VectorBuilder.

Techniques: Expressing, Staining, Labeling, Concentration Assay

Journal: PLOS Biology

Article Title: Phosphoinositide species and filamentous actin formation mediate engulfment by senescent tumor cells

doi: 10.1371/journal.pbio.3001858

Figure Lengend Snippet: (A ) Time course live-cell imaging of a senescent MCF-7 cell that expresses PLCD1-mCherry and P4M-SidMx2-GFP, throughout the entire process of engulfing NIR-MCF-7 prey cells in a DOXO-NT culture. Closed arrows indicate examples PLCD1-mCherry and P4M-SidMx2-GFP colocalization during engulfment. ( B ) Time course live-cell imaging of a senescent MCF-7 cell that expresses PLCD1-mCherry and 2xFYVE-GFP, throughout the entire process of engulfing a NIR-MCF-7 cell. Closed arrows indicate localization of PLCD1-GFP, but not 2xFYVE-mCherry, at mid stage engulfment. Open arrows indicate 2xFYVE-mCherry localization, but not PLCD1-GFP, at late-stage engulfment. Scale bar = 100 μm for A+B. ( C ) Volume view reconstruction of a senescent MCF-7 cell expressing PLCD1-GFP and P4M-SidMx2-mCherry that is engulfing 3 NIR-MCF-7 cells. Middle panels show separate color channels; bottom panel shows axial planes from bottom to top. Closed arrows indicate examples PLCD1-mCherry and P4M-SidMx2-GFP colocalization during engulfment, scale bar = 10 μm. ( D ) Volume view reconstruction of a senescent MCF-7 cell expressing PLCD1-mCherry and 2xFYVE-GFP, which is engulfing 2 NIR-MCF-7 cells. Middle panels show separate color channels; bottom panels show axial planes from bottom to top. Closed arrows indicate localization of PLCD1-mCherry, but not 2xFYVE-GFP, at mid stage engulfment. Open arrows indicate 2xFYVE-mCherry localization, but not PLCD1-GFP, at an internalized cell, scale bar = 10 μm.

Article Snippet: 2xFYVE-domain-mCherry fusion protein is lentiviral plasmid from VectorBuilder.

Techniques: Live Cell Imaging, Expressing

Journal: PLOS Biology

Article Title: Phosphoinositide species and filamentous actin formation mediate engulfment by senescent tumor cells

doi: 10.1371/journal.pbio.3001858

Figure Lengend Snippet: ( A ) CRISPR-Cas9 single-cell clone PIK3C2B knockouts of MCF-7 were screened by immunoblot. Clones indicated by asterisk were chosen for further testing. ( B ) Predator cell engulfment rates (left) and confluency as a measure of viability (right) for senescent MCF-7 parental cells and 3 PIK3C2B knockout clones were determined by time course imaging in a DOXO-NT culture. Underlying data can be found at . ( C ) Time course live-cell imaging of a senescent MCF-7 cell expressing 2xFYVE-mCherry and PIK3C2B-GFP throughout the entire process of engulfing an NIR-MCF-7 cell, scale bar = 100 μm. ( D ) Left: Merged (top) and separate (middle, lower) color channels of axial planes from bottom to top of a senescent MCF-7 cell expressing 2xFYVE-mCherry and PIK3C2B-GFP, engulfing NIR-MCF-7 cells. Right, volume view reconstruction of the same image, scale bar = 10 μm.

Article Snippet: 2xFYVE-domain-mCherry fusion protein is lentiviral plasmid from VectorBuilder.

Techniques: CRISPR, Western Blot, Clone Assay, Knock-Out, Imaging, Live Cell Imaging, Expressing